Continuous process for separation of proteins

ABSTRACT

Disclosed is a continuous process for separating or extracting proteins from a low grade mixture of a protein of interest, other proteins, impurities, and salts in a continuous simulated moving bed separation process. The invention provides for direct extraction of heme protein and plant protein from a crude mixture of such proteins, other proteins, impurities and salts using the chromatographic technique of simulated moving bed (SMB) continuous chromatography. The SMB process combines the steps of feed loading, adsorbent washing, product elution, adsorbent regeneration, and adsorbent equilibration. The novel strong anion exchange resin adsorbent is a quaternary amine cross-linked microcellulose wherein the microcellulose is cross-linked with epichlorohydrin and the quaternary amine is 2,3-epoxypropyltrimethyl-ammonium chloride which exhibits selective adsorption of proteins and complete regeneration. Purified protein separated in this manner may provide human health benefits by providing greater medicinal and nutrition opportunities from low quality protein sources.

FIELD OF THE INVENTION

The invention relates to a continuous method for separating orextracting proteins from a low grade mixture of proteins and impuritiesin a continuous direct separation process. More particularly, theinvention provides for direct extraction of a protein directly from amixture of proteins and impurities derived from plant protein extractusing chromatographic technique of simulated moving bed continuouschromatography, employing a selective adsorbent as the stationary phaseadsorbent, wherein the selective adsorbent is a strong anion exchangeresin. Most specifically, the strong anion exchange resin has a backbonematrix comprising a cross linked styrene divinyl benzene polymer, ahydroxylated polymethacrylate polymer, or a cross linkedmicrocrystalline cellulose resin. Purified protein separated in thecontinuous manner of the instant invention may provide human healthbenefits by providing greater medicinal and nutrition opportunities fromlow quality protein sources.

BACKGROUND

Proteins are important constituents of foods. Proteins are a majorsource of energy, and proteins contain essential amino-acids, such aslysine, tryptophan, methionine, leucine, isoleucine and valine, whichare essential to human health. Proteins are the major structuralcomponents of many natural foods, and a particular protein in a food candetermine the food structure or texture, e.g., tenderness of meat orfish products. Some individual proteins are used as food ingredientsbecause they can improve appearance, texture or stability. Suchindividual proteins are employed as gelling agents, emulsifiers, foamingagents, and thickeners.

There is an increasing requirement for protein production in academicand industrial settings for a variety of applications like drugdiscovery, biopharmaceutical production, and the food industry.Currently, large-scale production strategies are capable of providingcell cultures or fermentation titres containing 25-50 wt-% of thedesired protein, and the remaining 50-75 percent impurities. Currentapplications require that essentially all impurities be removed from ahighly purified protein product, and that the highly purified proteinproduct can be produced in commercial quantities.

Heme protein refers to a metallo protein which contains a reduced ironatom, Fe²⁺ in the center of a complex hydrophobic structure. Hemeproteins play a role in critical physiological functions because theiron atom in heme protein make heme proteins responsive to moleculeswhich can bind to divalent iron, such as oxygen, nitric oxide, carbonmonoxide, and hydrogen sulfide. Hemoglobin and myoglobin are types ofheme proteins which are essential for storing and transporting oxygen inmammals. Hemoglobin is also found in the root nodules of some plants.Leghemoglobin (legHb) occurs in the root nodules of leguminous plants,where it facilitates the diffusion of oxygen to symbiotic bacteriods inorder to promote nitrogen fixation.

Plant proteins, such as Ribulose-1,5-bisphosphate carboxylase oxygenase,most commonly known by the shorter name RuBisCO, is an plant enzymeinvolved in the Calvin cycle that catalyzes the first major step ofcarbon fixation, a process by which atmospheric carbon dioxide is madeavailable to organisms in the form of energy-rich molecules such asglucose. RuBisCO is very important in terms of biological impact,because it catalyzes the primary chemical reaction by which inorganiccarbon permanently enters the biosphere. RuBisCO is also the mostabundant protein in leaves, and is considered to be the most abundantprotein on Earth.

Cellulose is the main component of higher plant cell walls and one ofthe most abundant organic compounds on earth. It can be derived from anumber of sources using a number of techniques that are consideredsynthetic, and some that might be considered non-synthetic (natural). Itis available in many forms for different functional purposes in foodproducts. The three main forms of cellulose that have been consideredfor various uses are powdered cellulose, regenerated cellulose casing,and microcrystalline cellulose. Microcrystalline cellulose is purified,partially depolymerized cellulose. It is a fine, white, odorlesscrystalline powder which is insoluble in water, insoluble in diluteacids, insoluble in most organic solvents, and also insoluble in dilutesodium hydroxide solutions. Microcrystalline cellulose is primarily usedin food to stabilize and improve the body, texture, and stability offood products.

Ion exchangers which have been used in separating whey proteins fromwhey protein containing solutions include both cation exchangers,particularly of the SP or SE (sulphonate) or CM (carboxymethyl) type,and anion exchangers, particularly of the QA (quaternary amino) or DEAE(diethylaminoethyl) type. In terms of the exchanger matrix itself, manyinsoluble matrices have been used, including cellulose, cross-linkeddextran, cross-linked agarose, synthetic hydophilic polymers andinorganic materials coated with hydrophilic polymers. One matrix thathas proved to be particularly useful in large scale separation andpurification of whey proteins is regenerated cellulose which has beenhydroxyalkylated and cross-linked. Ion exchangers prepared on thismatrix are resistant to attrition, have high protein capacity, high flowproperties and are available at relatively low cost.

Preparation of quaternized celluloses is known in the art, for examplefrom U.S. Pat. No. 3,472,840 assigned to Union Carbide Corporation,which discloses cellulose derivatives, particularly cellulose etherscontaining quaternary ammonium groups which are used in the many fieldsin which cellulose ethers cannot be employed.

U.S. Pat. No. 6,911,483 to Ayers et al. discloses an anion exchangercomprising a water insoluble, hydrophilic, water swellable,hydroxy(C₂-C₄ alkylated and cross-linked regenerated cellulose,derivatized with quaternary amino (QA) groups. Ayers et al. disclosesand claims that such anion exchangers have a level of substitution ofthe QA groups of 1.4 milliequivalents per dry gram of anion exchanger(meq/g) or greater. Anion exchangers are useful for separating proteinsfrom protein-containing solutions, and particularly for adsorbing wheyproteins from whey protein-containing solutions. Ayers et al. furtherdifferentiates such anion exchanger compositions based on regeneratedcellulose from materials produced from microcrystalline cellulose withreference to Antal et al. Antal et. al. (Carbohydrate Polymers 19,167-169, 1992) describe the optimization of the reaction ofmicrocrystalline cellulose with the alkylating agentsCHPTAC-(chloro-2-hydroxypropyl)trimethylammonium chloride) and1,3-bis(3-chloro-2-hydroxy-propyl)imidazolium hydrogen sulfate inalkaline medium. Ayers et al. states that the maximum substitution levelthey [Antal et al.] were able to obtain with CHPTAC was 0.94 meq/g(mmol/g, millimoles per gram)), although the second reagent gave aproduct with 1.56 meq/g. No protein capacities are given and it islikely that the latter reagent, being bifunctional, would haveintroduced extensive crosslinking into the cellulose to the detriment ofprotein capacity. Furthermore, Ayers et al. states that microcrystallinecellulose is not a suitable matrix for repeated use on a largeindustrial scale.

Fibrous cellulose has been derivatized with quaternary ammonium groupsto a high degree of substitution, DS of at least 0.5 (>2 meq/g), using avery large excess of alkylating reagent containing quaternary ammoniumgroups. The cellulose is either not crosslinked (1998 U.S. Pat. No.5,731,259) or crosslinked (1998 U.S. Pat. No. 5,780,616). Preferably thealkylating reagent is used in 20:1 to 40:1 mole ratio of reagent toanhydroglucose units of cellulose. In the case of GTAC this amounts to186-372 g of reagent per 10 g of cellulose used either in 5-8 repeatedreactions or one large addition of the solid reagent with 30 mL ofwater. The products, described at one point as a jelly mass, are usefulas superabsorbents for water and saline solutions in the field ofhygenic-sanitary products such as diapers for babies. They are designedto be used once and then disposed of and are not at all suitable forrepeated use day after day in a reactor or column bed where physicalrobustness against attrition, long life and high flow-through rates arerequired for anion exchangers processing protein solutions.

Processes are sought for the large scale separation and continuouspurification of proteins, such as heme proteins and plant proteins, frommixtures of protein-containing solutions such as animal and vegetablederived protein extracts.

SUMMARY

The process of the present invention relates to the purification ofproteins directly from mixtures of protein containing solutions usingnovel chromatographic techniques. More specifically, Applicant hasdeveloped a novel simulated moving bed separation process (SMB) seriesof adsorbent/desorbent combinations and SMB configurations to bringabout the enrichment and purification of protein from mixtures ofprotein containing solutions directly from such mixtures withoutbreaking down the protein and without extreme chemical and physicalseparation conditions such as heat or the use of supercritical pressure.Options for the SMB stationary phase adsorbent include strong anionexchange resin or a novel adsorbent based on microcrystalline cellulose.The novel SMB adsorbent developed for the SMB process of the inventioncomprises a cross-linked microcrystalline cellulose/quaternary amino(QA) anion exchanger as a stationary phase adsorbent in combination witha series of buffer desorbents to provide an enriched extract stream richin purified protein, and a series of waste raffinate streams comprisingimpurities. A purified protein product having a purity greater than 80wt percent (e.g., 82, 83, 85 wt-%) following solvent removal can beobtained.

In one embodiment, the invention is a process for the continuousextraction of heme protein from an aqueous protein mixture comprisingheme protein, water, other proteins, contaminants and salts in of acontinuous simulated moving bed (SMB) extraction unit. The continuousSMB extraction unit has a plurality of n adsorbent beds, and eachadsorbent bed has a sequence number j from 1 to n and is functionallydisposed in a capture zone, a feed zone, a washing zone, an elutionzone, a regeneration zone, and an equilibration zone. Each of the zonesis sequentially disposed in parallel from the capture zone beingnumbered as a first adsorbent bed to the equilibration zone having alast or n^(th) adsorbent bed. Each of the feed zone, the washing zone,the elution zone, the equilibration zone, and the capture zone containsone or more adsorbent beds, and each adsorbent bed has a top and abottom. Each adsorbent bed contains an adsorbent comprising a quaternaryamine cross-linked microcrystalline resin. The process of continuousextraction of heme protein comprises the following steps:

-   a) concurrently passing the aqueous protein mixture at SMB feed    conditions to the top of the feed zone to load the aqueous protein    mixture on the adsorbent in the feed zone and withdrawing a first    waste stream comprising water and contaminants from the bottom of    the feed zone;-   b) concurrently passing a wash buffer stream to the top of the wash    zone to wash the adsorbent in the wash zone to provide a feed purge    stream and withdrawing the feed purge stream from the bottom of the    wash zone;-   c) concurrently passing at least a portion of the feed purge stream    to the top of the capture zone having the first adsorbent bed to    capture any unbound protein of interest in the capture zone and    provide a first waste stream and withdrawing the first waste stream    from the bottom of the capture zone;-   d) concurrently passing an elution buffer to the top of the elution    zone and eluting a protein extract product comprising heme protein    from the bottom of the elution zone;-   e) concurrently or counter-currently passing a first regeneration    buffer comprising a salt and a base to a first portion of the    regeneration zone and simultaneously passing a second regeneration    buffer comprising an acid to a second portion of the regeneration    zone disposed in parallel to the first portion of the regeneration    zone and withdrawing a second waste stream from the first and second    portions of the regeneration zones;-   f) concurrently passing an equilibration buffer to the top of the    equilibration zone having the last adsorbent bed to restore ionic    activity to the adsorbent in the equilibration zone and withdrawing    a third waste stream from the bottom of the equilibration zone; and,    indexing the adsorbent beds wherein the first adsorbent bed in the    capture zone is advanced to the feed zone and the last adsorbent bed    is advanced to the capture zone to provide a continuous process.

In another embodiment, the invention is a process for the continuousextraction of at least one protein of interest from a crude protein in acontinuous simulated moving bed (SMB) extraction zone. The processcomprises:

-   -   a) admixing a crude protein comprising the at least one protein        of interest, other proteins, and impurities with an        equilibration buffer stream comprising a phosphate salt of        sodium or potassium and water to provide an SMB feed stream:    -   b) continuously passing the SMB feed stream at SMB feed        conditions and a plurality of wash streams to the continuous        simulated moving bed (SMB) extraction zone, the continuous SMB        extraction zone having a plurality of adsorbent beds, one or        more of the adsorbent beds being disposed in a loading zone, a        washing zone, a elution zone, a regeneration zone, an        equilibration zone, and optionally a capture zone or a second        washing zone, each of the zones being sequentially disposed in        parallel from the loading zone having a first adsorbent bed in a        first position to the equilibration zone having a last adsorbent        bed in a last position, each of the loading zone, the washing        zone, the elution zone, the equilibration zone, and the        equilibrium zone containing the one or more adsorbent beds, each        of the one or more adsorbent beds having a top and a bottom and        each of the one or more adsorbent beds containing an adsorbent        selected from the group consisting of a strong anion exchange        resin and a quaternary amine cross linked microcrystalline        resin, said plurality of wash streams having a pH of from about        7.9 to about 8.2 including a wash buffer stream, an elution        buffer stream, a regeneration buffer stream, and the equilibrium        buffer stream, said continuous SMB extraction zone comprising:        -   i) concurrently passing the SMB feed stream at SMB feed            conditions to the top of the loading zone containing a first            adsorbent bed to load the SMB feed mixture on the adsorbent            in the loading zone and withdrawing a first waste stream            comprising water and impurities from the bottom of the            loading zone;        -   ii) concurrently passing the wash buffer stream comprising            phosphate salt of sodium or potassium to the top of the            washing zone to wash the adsorbent in the wash zone to            provide a wash effluent stream and withdrawing the wash            effluent stream from the bottom of the wash zone;        -   iii) concurrently passing the elution buffer comprising            phosphate salt of sodium or potassium and chloride salt of            sodium or potassium to the top of the elution zone and            eluting an elution effluent stream comprising the protein of            interest from the bottom of the elution zone;        -   iv) concurrently or counter-currently passing the            regeneration buffer comprising phosphate salt of sodium or            potassium and a base to the regeneration zone and            withdrawing a second waste stream from the regeneration            zone;        -   v) concurrently passing the equilibration buffer comprising            phosphate salt of sodium or potassium and optionally a            chloride salt of sodium or potassium to the top of the            equilibration zone comprising the last adsorbent bed to            restore ionic activity to the adsorbent in the equilibration            zone and withdrawing a third waste stream from the bottom of            the equilibration zone; and,        -   vi) indexing the adsorbent beds wherein the first adsorbent            bed in the loading zone is advanced by one adsorbent bed            toward the equilibration zone and the last adsorbent bed in            the equilibration zone is repositioned to the first position            in the loading zone to provide the continuous SMB process;    -   c) passing the first waste stream, the second waste stream, the        third waste stream, and at least a portion of the wash effluent        stream to waste disposal; and,    -   d) recovering the elution effluent stream as a protein product        stream comprising the protein of interest.

In a still further embodiment, the invention is an adsorbent for use inchromatographic separation and extraction of protein. The adsorbentcomprises a microcrystalline cellulose which has been cross linked withepichlorohydrin and reacted with 2,3-epoxypropyltrimethyl-ammoniumchloride

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic process flow diagram of the simulated moving bedseparation process illustrating one embodiment of the invention.

FIG. 2 is a schematic process flow diagram of the simulated moving bedseparation process illustrating one embodiment of the invention using 15adsorbent beds.

FIG. 2a is a schematic process flow diagram of the simulated moving bedseparation process illustrating one embodiment of the invention using 15adsorbent beds.

FIG. 3 is an area plot of the composition of the eluate fractionswithdrawn from the protein purification process of one embodiment of theinvention for heme protein using a cross linked microcelluloseadsorbent.

FIG. 4 is a gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced for hemeprotein using a cross linked microcellulose adsorbent by one embodimentof the invention.

FIG. 5 is an area plot of the composition of the eluate fractionswithdrawn from the protein purification process of one embodiment of theinvention for purifying heme protein using a strong anion exchangeresin.

FIG. 6 is a gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced by oneembodiment of the invention for purifying heme protein using a stronganion exchange resin.

FIG. 7 is an area plot of the composition of the eluate fractionswithdrawn from the protein purification process of one embodiment of theinvention for purifying RuBisCO on a strong anion exchange resin.

FIG. 8 is a gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions for purifying RuBisCOproduced by one embodiment of the invention using a strong anionexchange resin.

FIG. 9 is a schematic process flow diagram of the simulated moving bedseparation process illustrating one embodiment of one embodiment of theinvention for extracting RuBisCO.

FIG. 10 is a schematic process flow diagram of the simulated moving bedseparation process illustrating one embodiment of the invention forextracting pea proteins.

FIG. 11 is an area plot of the composition of the eluate fractionswithdrawn from the protein purification process of one embodiment of theinvention for purifying pea proteins on a strong anion exchange resin.

FIG. 12 is a gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions for purifying peaproteins produced by one embodiment of the invention using a stronganion exchange resin.

FIG. 13 is a schematic block flow diagram of an 8 bed simulated movingbed process of Example 6 for purification of leghemoglobin.

FIG. 14 shows the optical density of the elute fractions withdrawn fromthe Simulated Moving Bed process in Example 6.

FIG. 15 is gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Proteins are complex ampholytes that have both positive and negativecharges. Ionic interactions are the basis for purification of proteinsby ion exchange chromatography. Ion exchange chromatography is one ofthe most powerful protein purification technique and most frequentlyused chromatographic technique used for the separation of proteins. Theseparation is due to competition between proteins with different surfacecharges for oppositely charged groups on an ion exchange adsorbant. Theproperties of the ion exchanger will also influence the separation. Thestructural backbone of the ion exchange resin can be made up ofdifferent types of polymer backbones, fixed groups with differentchemistries.

Ion exchange chromatography relies on the reversibleadsorption-desorption of ions in solution to a charged solid matrix orpolymer network. This technique is the most commonly usedchromatographic technique for protein separation. A positively chargedmatrix is called an anion-exchanger because it binds negatively chargedions (anions). A negatively charged matrix is called a cation-exchangerbecause it binds positively charged ions (cations). The bufferconditions (pH and ionic strength) are adjusted to favor maximum bindingof the protein of interest to the ion-exchange column. Contaminatingproteins, which bind weakly will pass more rapidly through the columnwhile those bind strongly will elute at higher concentrations of saltcompared to the protein of interest. The protein of interest is theneluted using another buffer solution which favors its desorption at aspecific salt concentration from the column (e.g., different pH or ionicstrength).

Stationary Phase

The stationary phase for use in the continuous simulated moving bedprocess of the present invention comprises or contains a strong anionexchange resin which has a backbone matrix comprising a cross linkedstyrene divinyl benzene polymer, a hydroxylated polymethacrylatepolymer, or a cross linked microcrystalline cellulose resin. Morespecifically, Applicant developed a cross linked microcrystallinecellulose resin with quaternary amine which provided a stable and easilyregenerated stationary phase for SMB operation. An example of a stronganion exchange resin having a backbone matrix comprising a cross linkedstyrene divinyl benzene polymer include DIAION HPA25L, a strongly basicanion which represented a strong anion exchange resin with exchangeresin (Available from Mitsubishi Chemical Company, Tokyo, Japan). Anexample of a strong anion exchange resin having a backbone matrixcomprising a hydroxylated polymethacrylate polymer is TOYOPEARL GIGACAPQ-650, a high capacity, high resolution, strong anion exchange resin(Available from Tosoh Bioscience LLC, King of Prussia, Pa.). Thesestrong anion or strong basic exchange resins provided a selectiveadsorption of the protein of interest from a mixture of the protein ofinterest in dilute aqueous mixtures and in the presence of impuritiessuch as lipids, color impurities. A key property of the adsorbents ofthe present invention is the ability of the adsorbent to be regeneratedwithin the operation of cycle of the SMB process and restore theadsorbent to its initial adsorption activity. Applicant discovered anovel adsorbent prepared by crosslinking a microcrystalline cellulosewith epichlorohydrin and exchanging the cross linked microcrystallinecellulose with a quaternary amine (2,3-epoxypropyltrimethyl-ammoniumchloride (glycidyltrimethylammonium chloride) to provide a quaternaryamine cross-linked microcrystalline resin having a backbone matrixcomprising a cross linked microcrystalline cellulose resin.

Mobile Phase Desorbent

Unlike a traditional SMB process which uses a single mobile phasedesorbent to sweep the adsorbent in the adsorbent beds, the presentinvention employs a series of different desorbent buffer solutions indifferent portions of the process to facilitate the steps of the SMBprocess. For example, the SMB process of the present invention comprisesa feed step, a wash step, an elution step, a regeneration step, and anequilibration step. All of these steps are carried out in a repeatingparallel sequence, in a manner which allows the continuous flow of feedto the SMB process and the continuous production of the protein product.Also produced by the process are a number of waste steams which areaqueous streams and represent spent buffer solutions which may containsalts, unbound/bound proteins or protein fragments, and small molecules.The waste streams generated or withdrawn from the SMB may be neutralizedand disposed of in a conventional manner. By way of example for theextraction of heme protein, such as leghemoglobin, from yeast lysate, inthe wash step, a wash buffer comprising potassium phosphate,concentration of 2-10 mM and having a pH of about 7.5 to 8.5 and aconductivity of between 0.5 and 2 mS/cm. More preferably, the washbuffer has a potassium phosphate concentration of 3-6 mM. In the elutionstep, an elution buffer comprising potassium phosphate and sodiumchloride and having a pH of 7.5-8.5 and a conductivity of 5-10 mS/cm isemployed to elute the protein from the adsorbent and recover the proteinproduct. In the regeneration step, preferably two regeneration buffersolutions are employed to regenerate the adsorbent and reestablish theionic balance of the adsorbent. A first regeneration buffer comprisingsodium hydroxide and sodium chloride is employed to remove tightly boundcontaminants from the adsorbent and a second regeneration buffer isemployed as an acid wash to restore the activity of the adsorbent. Thesecond regeneration buffer comprises a dilute solution of hydrochloricacid (HCl) or phosphoric acid (H₃PO₄). The first regeneration buffercomprises a concentration of sodium hydroxide of about 0.1 N-2 M NaOHand a concentration of sodium chloride of about 0.5-2M NaCl. The secondregeneration buffer has a concentration of hydrochloric acid of about0.1 N-0.2N HCl or 0.1 N-0.2N H₃PO₄. In the equilibration step, anequilibration buffer was employed to return the adsorbent to itsoriginal ionic strength by contacting the adsorbent with an aqueoussolution containing potassium phosphate. It was discovered that to carryout the SMB process wherein the above mentioned major steps of theprocess could be carried out in essentially equal step times within theSMB cycle, that the equilibration step must be carried out in at leasttwo stages. In the first equilibration stage, a first equilibrationbuffer comprising potassium phosphate and having a pH of about 7.5 to8.5 was passed through the adsorbent bed following the regenerationstep. The first equilibration buffer had a potassium phosphateconcentration of about 50-200 mM potassium phosphate at the required pH.In the second equilibration step the second equilibration buffer was a2-10 mM solution of potassium phosphate having a pH of between about 7.5and 8.5.

Feed Preparation

In the present invention, the feed can be a yeast extract such aslysate, or crude protein, admixed with water, such as deionized water oran equilibration buffer to provide an SMB feed mixture. The crude feedmixture may comprise a protein of interest such as the heme protein,leghemoglobin, as well as other proteins, protein fragments, smallmolecules, and salts. The concentration of total protein in the feedmixture comprise or contain between about 1.5 to about 10 grams of crudeprotein per liter of feed mixture. It is also important to preventdegradation of the feed by maintaining the temperature of the feedmixture at an SMB feed temperature which is at or below about 4° C. andto maintain a feed pH of between about 7.5 and about 8.5. Morepreferably the pH of the feed mixture should be maintained at about 8.0.

DETAILED DESCRIPTION OF THE DRAWINGS

With reference to FIG. 1, the following adsorbent bed organizationrelates to a simulated moving bed (SMB) separation process for thecontinuous separation of heme proteins from a crude feed mixturecomprising yeast and water. As shown in FIG. 1, in a simplified form,the simulated moving bed of the present invention comprises 6 adsorptionzones: Capture zone 31, Feed Loading Zone 32, Wash Zone 33, Elution Zone34, Regeneration Zone A35 and Regeneration Zone B 36, and EquilibrationZone 37. Each of the 6 adsorption zones may comprise one or moreadsorbent columns arranged in parallel, wherein each adsorbent column isfilled with or contains and adsorbent consisting of a strong anionexchange resin or a quaternary amine cross-linked microcrystalline resinfor the concentration and recovery of heme protein in the crude feedmixture. The crude feed mixture comprises an aqueous solution orsuspension of yeast lysate in a feed concentration of from about 1.5 to10 grams of crude protein, such as lysate, per liter. More preferably,the crude feed mixture comprises a feed concentration of from about 1.5to 10 grams of crude protein per liter. The crude feed mixture ismaintained at an SMB feed temperature at or below about 4° C. and pHadjusted to have a pH of between about 7.5 to about 8.5 and an a feedelectrical conductivity, or feed specific conductance, of about 0.5 toabout 2.0 mS/cm (milliSiemens/centimeter), and more preferably a feedspecific conductance of about 0.8 to about 1.5 mS/cm(milliSiemens/centimeter). In a given SMB cycle, the feed mixture inline 10 is passed to the top of feed loading zone 32 and therein thefeed mixture is contacted with the adsorbent at a feed pH of 7.5 toabout 8.5 and a feed temperature of less than or equal to about 4° C.The crude feed mixture is passed through the feed loading zone 32 in aconcurrent direction; that is, from the top of the feed loading zone tothe bottom of the feed loading zone. As the feed mixture in line 10 isloaded on the feed loading zone 32, a first waste stream in line 12 iswithdrawn from the feed loading zone 32. The first waste stream in line12, comprising water and unbound protein may be neutralized and disposedof in any conventional manner. A wash buffer in line 14 is passed to thewash zone 33 in a concurrent direction from the top of the wash zone tothe bottom of the wash zone and a second waste stream is withdrawn inline 16. At least a portion of the second waste stream in line 16 isemployed to concurrently purge the capture zone 31 to prepare thecapture zone 31 for the introduction of the feed mixture and a thirdwaste stream is withdrawn in line 1. An elution buffer in line 18 isconcurrently passed to the top of the elution zone 34 and a proteinproduct stream in line 20 is withdrawn from the bottom of the elutionzone 34. A first regeneration buffer in line 22 comprising a sodium baseand a sodium salt is passed counter currently to the bottom ofregeneration zone A 35 to remove any tightly bound contaminants from theadsorbent and simultaneously a second regeneration buffer comprising anacid (such as HCl or H₃PO₄) is passed counter-currently to regenerationzone B to acid wash the adsorbent and a fourth waste stream is withdrawnfrom the tops of regeneration zones A (35) and B (36) in line 24. Thefourth waste stream in line 24 may be neutralized and passed to wastedisposal in a conventional manner. Regeneration of the regenerationzones 35 and 36 with countercurrent passing of the regeneration buffersassures the removal of impurities which buildup at the top of theadsorbent columns in the regeneration zones during the operation of theSMB process. An equilibration buffer in line 26 is passed concurrentlyto the top of the equilibration zone 37 to equilibrate the adsorbent anda fifth waste stream is withdrawn in line 28. The fifth waste stream maybe passed to neutralization and waste disposal, or alternativelyrecycled after pH adjustment and filtration to offset the amount of theequilibration buffer in line 26. Each of the above steps takes placeduring each time period of the SMB cycle. At the end of the period, thecycle is indexed or incremented by shifting the all or a portion of eachof the 6 adsorption zones one increment to the left wherein one or moreof the adsorption beds in the equilibration zone 37 is moved to thecapture zone 31. To accomplish this shift, a rotary valve or a valveswitching controller permits the beds to be shifted in function withoutphysically moving the adsorption beds between the zones, or theadsorption zones may be physically rotated about a valve manifold on acarousel which accomplishes the incrementing or shifting of theadsorption beds to the left, or counter to the direction of the mobilephase.

In another embodiment of the invention is illustrated using 15adsorption beds in FIG. 2 for simulated moving bed (SMB) separationprocess for the continuous separation of heme proteins (leghemoglobin)from a feed mixture comprising yeast lysate and water. As shown in FIG.2, the simulated moving bed of the present invention comprises 15adsorption beds (V1-V15) disposed in adsorption zones: Capture Zone(V1), Feed Loading Zone (V2-V3), Wash Zone (V4-V5), Elution Zone(V6-V9), First Regeneration Zone A (V10-V11) and Second RegenerationZone B (V12), and Equilibration Zone (V13-V15). Each adsorbent bed has atop and a bottom. The individual adsorption beds (V1-V15) and arearranged serially from left to right and grouped in the fivefunctionally isolated segments. Each isolated segment comprises at leastone or more of the adsorbent beds. In each isolated segment, with theexception of the regeneration zone, an external stream is introduced tothe top of a first adsorbent bed in the isolated segment and a wastestream or a product stream is withdrawn from the bottom of the lastadsorbent bed in sequence serially, from left to right. In theregeneration zone of the process scheme shown in FIG. 2, theregeneration zone adsorbent beds are processed in a counter currentmanner, wherein the external streams are introduced at the bottom of theadsorbent bed, and the effluent is withdrawn from the top of theadsorbent bed. Each of the adsorbent beds contains a stationary phaseadsorbent which is selective for the adsorption of protein as describedhereinabove. According to FIG. 2, a protein feed stream in line 90, suchas a crude lysate stream comprising crude lysate and water, is splitinto a first feed portion in line 92 and a second feed portion in line91. The first feed portion in line 92 is introduced to the top ofadsorbent bed V2 and a first waste effluent in line 94 is withdrawn fromadsorbent bed V2. The second feed portion in line 91 is introduced tothe top of adsorbent bed V3, operating in parallel to adsorbent bed V2,and a second waste effluent stream is withdrawn in line 93 from thebottom of adsorbent bed V3. A feed buffered wash stream in line 80 isintroduced to the top of adsorbent bed V1 to prepare adsorbent bed V1for processing the feed stream and a third waste effluent stream iswithdrawn from adsorbent bed V1 in line 96. The feed buffered stream inline 80 comprises a 2-10 mM sodium phosphate solution and has a pHranging from 7.5 to 8.5, preferably a pH of 8.0, and a conductivity offrom about 0.5-2.0 mS/cm. The first waste effluent stream in line 94,the second waste effluent stream in line 93, and the third wasteeffluent stream in line 96 are admixed to provide a first waste streamin line 95. The first waste stream is passed to waste disposal. A washbuffer stream in line 75 is split into a wash buffer “A” portion in line77 and a wash buffer “B” portion in line 76. The wash buffer stream inline 75 comprises a 2-10 mM sodium phosphate solution and has a pH of7.5 to 8.5 and a conductivity of from about 0.5-2.0 mS/cm. The washbuffer A portion in line 77 is introduced to the top of adsorbent bed V4and a first spent wash buffer is withdrawn in line 79. The wash buffer Bportion in line 76 is introduced to the top of adsorbent bed V5 and asecond spent wash buffer is withdrawn in line 78. The first spent washbuffer in line 79 and the second spent wash buffer in line 78 areadmixed to form the feed buffered stream in line 80. An elution bufferin line 65 comprises a mixture of a 2-10 mM solution of potassiumphosphate and a 25-60 mM solution of sodium chloride and has a pH offrom 7.5 to 8.5, preferably a pH of 8.0, and a conductivity of 3-10mS/cm, and is at room temperature of about 25° C. The elution buffer inline 65 is split into an elution buffer A in line 67 and an elutionbuffer B in line 68. The elution buffer “A” in line 67 is introduced orpassed to the top of adsorbent bed V8 and an intermediate eluate “A” iswithdrawn from the bottom of adsorbent bed V8 in line 69. Similarly,elution buffer “B” in line 67 is introduced or passed to the top ofadsorbent bed V9 and an intermediate eluate “B” is withdrawn from thebottom of adsorbent bed V9 in line 68. The intermediate eluates A and Bare admixed or combined in line 70 as a combined intermediate eluate andagain split into a first intermediate eluate in line 72 and a secondintermediate eluate in line 71. The first intermediate eluate is passedto the top of adsorbent bed V6 and a first protein product stream iswithdrawn in line 74. Similarly, the second intermediate eluate ispassed to the top of adsorbent bed V7 and a second protein productstream is withdrawn in line 73. The enriched protein product stream canbe characterized as having at least 75 wt-% protein purity and having aprotein content of ˜0.5 g/L. By the term protein purity it is meant thatin 100 g of total purified protein, 75 g is the protein of interest.Adsorbent beds V10-V12 comprise the regeneration zone. In theregeneration zone, the regeneration process comprises eitherconcurrently passing (See FIG. 2a ), or counter currently passing (SeeFIG. 2) separate regeneration buffers to separate portions of theregeneration zone. The regeneration zone is divided into a firstregeneration zone comprising adsorbent beds V11 and V10, and a secondregeneration zone comprising adsorbent bed V12. With reference to acounter current passing of the regeneration buffers through theregeneration zone, in the first regeneration zone, a first regenerationbuffer comprising a mixture of a 0.1-2 Normal (N) sodium hydroxide and0.5-2 Molar (M) solution of sodium chloride in water in line 59 isintroduced to the bottom of adsorbent bed V11 and a first regenerationintermediate stream is withdrawn from the top of adsorbent bed V11 inline 60. The first regeneration buffer counter currently flushes theadsorbent beds in the first regeneration zone to remove essentially allof undesired tightly bound proteins and other contaminants, such aslipids, color impurities which have concentrated in layers toward thetop of the adsorbent beds V10, V11, and V12 during the SMB process. Thefirst regeneration intermediate stream is passed to the bottom ofadsorbent bed V10 and a first regeneration waste stream is withdrawn inline 64. In the second regeneration step, a second regeneration bufferstream in line 58 comprising a 0.1-0.2N solution of hydrochloric acid isintroduced to the bottom of adsorbent bed V12 and a second regenerationwaste stream is withdrawn in line 61. The second regeneration buffercounter currently flushes the second regeneration zone of any remainingundesired contaminants and acid washes the adsorbent bed V12 to restoreinitial activity to the stationary phase adsorbent. The first and secondregeneration waste streams in lines 61 and 64 are admixed or combined inline 63 to provide a combined regeneration waste stream. Theregeneration waste stream in line 63 is passed to waste disposal. Theequilibration zone comprises a first equilibration zone as adsorbent bedV13, and a second equilibration zone as adsorbent beds 14 and 15. Afirst equilibration buffer in line 57 comprising a solution of 50-200 mM(milliMolar) sodium phosphate and having a pH of about 8 is passed tothe top of adsorbent bed 13 and a first equilibration waste stream iswithdrawn from the bottom of adsorbent bed V13 on line 55. A secondequilibration buffer in line 50 comprising a 4 mM solution of potassiumphosphate to provide the final equilibration of the stationary phaseadsorbent is split equally into lines 52 and 51 and passed to the top ofadsorbent beds V14 and V15, respectively. A second equilibration wastestream in line 54 is withdrawn from adsorbent bed V14, and a thirdequilibration waste stream in line 53 is withdrawn from adsorbent bedV15. The first and second and third equilibration waste streams arecombined or admixed in line 56 to provide a combined equilibration wastestream. It was discovered that by dividing the equilibration step into afirst equilibration at high concentration of potassium phosphate (50-200mM solution) followed by a second or final equilibration step using alower concentration of potassium phosphate (2-10 mM solution),sufficient equilibration of the stationary phase adsorbent can becarried out within a single SMB cycle.

With reference to FIG. 2a , a further embodiment of the invention isillustrated using 15 adsorption beds for simulated moving bed (SMB)separation process for the continuous separation of proteins from a feedmixture comprising yeast lysate and water as described hereinabove inFIG. 2, except that the regeneration zone is operated in a concurrentmode. The description of FIG. 2a is identical to the description of FIG.2, with the exception of that operation of the regeneration zone(V10-V12) takes place in a concurrent manner. In the concurrent mode inthe regeneration zone of FIG. 2a , the external streams are introducedat the top of the adsorbent beds and the effluent is withdrawn from thebottom of the adsorbent beds (V10-V12) in the regeneration zone. Asshown in FIG. 2a , the simulated moving bed of the present inventionagain comprises 15 adsorption beds (V1-V15) disposed in adsorptionzones: Capture Zone (V1), Feed Loading Zone (V2-V3), Wash Zone (V4-V5),Elution Zone (V6-V9), First Regeneration Zone A (10-V11) and SecondRegeneration Zone B (V12), and Equilibration Zone (V13-V15). Eachadsorbent bed has a top and a bottom. The individual adsorption beds(V1-V15) and are arranged serially from left to right and grouped in thefive functionally isolated segments. Each isolated segment comprises atleast one or more of the adsorbent beds. In every isolated segment, anexternal stream is introduced to the top of a first adsorbent bed in theisolated segment and a waste stream or a product stream is withdrawnfrom the bottom of the last adsorbent bed in sequence serially, fromleft to right. In the concurrent regeneration zone, the regenerationprocess comprises concurrently passing separate regeneration buffers toseparate portions of the regeneration zone. The regeneration zone isdivided into a first regeneration zone comprising adsorbent beds V11 andV10, and a second regeneration zone comprising adsorbent bed V12. Withreference to the concurrent passing of the regeneration buffers throughthe regeneration zone of FIG. 2a , in the first regeneration zone(V10-V11), a first regeneration buffer comprising a mixture of a 0.1-2MNormal (N) sodium hydroxide and 0.5-2 Molar (M) solution of sodiumchloride in water in line 59 is introduced to the top of adsorbent bedV11 and a first regeneration intermediate stream is withdrawn from thebottom of adsorbent bed V11 in line 60. The first regeneration bufferconcurrently flushes the adsorbent beds in the first regeneration zoneto remove essentially all of undesired tightly bound proteins and othercontaminants, such as lipids, color impurities which have concentratedin adsorbent beds V10, and V11 during the SMB process. The firstregeneration intermediate stream in line 60 is passed to the top ofadsorbent bed V10, and a first regeneration waste stream is withdrawn inline 64. In the second regeneration step, a second regeneration bufferstream in line 58 comprising a 0.1 N solution of hydrochloric acid isintroduced to the top of adsorbent bed V12, and a second regenerationwaste stream from the bottom of adsorbent bed V12 is withdrawn in line61. The second regeneration buffer concurrently flushes the secondregeneration zone of any remaining undesired contaminants and acidwashes the adsorbent bed V12 to restore initial activity to thestationary phase adsorbent. The first and second regeneration wastestreams in lines 61 and 64 are admixed or combined in line 63 to providea combined regeneration waste stream. The regeneration waste stream inline 63 is passed to waste disposal. The remainder of the elements(adsorbent beds and streams) of FIG. 2a are identical to the processscheme described hereinabove in FIG. 2.

In a further embodiment of the invention is illustrated using 15adsorption beds in FIG. 9 for simulated moving bed (SMB) separationprocess for the continuous separation of a plant protein, RuBisCOprotein, from spinach extract. The feed mixture comprising the spinachextract diluted or dispersed in water or an equilibration buffer streamcomprising sodium or potassium or sodium phosphate and sodium orpotassium chloride in water. As shown in FIG. 9, the simulated movingbed comprises 15 adsorption beds (R1-R15) disposed in adsorption zones:Water Wash Zone (R15), Regeneration Zone (R14-R13), Elution Zone(R12-R10), Washing Zone (R9-R7), Feed Loading Zone (R6-R4), andEquilibration Zone (R3-R1). Each adsorbent bed has a top and a bottom.The individual adsorption beds (R1-R15) and are arranged serially fromleft to right and grouped in the above six functionally isolatedsegments. R1 is in a first position and R15 is in the last position.Each isolated segment comprises at least one or more of the adsorbentbeds. In each isolated segment an external stream is introduced to thetop of a first adsorbent bed in the isolated segment and a waste streamor a product stream is withdrawn from the bottom of the last adsorbentbed (lower number adsorbent bed) in sequence, serially, from left toright. Each of the adsorbent beds contains a stationary phase adsorbentwhich is selective for the adsorption of protein as describedhereinabove. According to FIG. 9, an SMB feed stream comprising thecrude protein in line 205 having a spinach concentration of about 0.5-70g/I is introduced to the top of adsorbent bed R6 in the feed loadingzone (R6-R4). The adsorbent beds R6, R5, and R4 are arranged in series,such that the R6 effluent stream withdrawn from the bottom of adsorbentbed R6 in line 216 is passed to the top of adsorbent bed R5, and the R5effluent stream in line 217 is passed to the top of adsorbent bed R4. Inthe feed loading zone the protein of interest is disposed on thestationary phase adsorbent in adsorbent beds R6, R5, and R4 and a firstwaste stream is withdrawn from adsorbent bed R4 in line 218 and passedto neutralization and waste disposal. A wash buffer stream in line 204comprising sodium or potassium phosphate and sodium or potassiumchloride is passed to the top of adsorbent bed R9 in a washing zone(R9-R7) to wash impurities and other proteins from the adsorbent in thewashing zone and provide a second waste stream in line 215. The washbuffer stream comprises a wash buffer concentration of from about 2 mMto about 100 mM of phosphate salt of sodium or potassium, and the washbuffer stream has a pH of about 7.5 to 8.5 and a conductivity of between0.5 and 25 mS/cm. The adsorbent beds R9, R8, and R7 are arranged inseries, such that the R9 effluent stream withdrawn from the bottom ofadsorbent bed R9 in line 213 is passed to the top of adsorbent bed R8,and the R8 effluent stream in line 214 is passed to the top of adsorbentbed R7. An elution buffer stream in line 203 comprising sodium orpotassium phosphate and sodium or potassium chloride and having aconcentration of 2 mM to about 100 mM of phosphate salt of sodium orpotassium, and from about 0.02 M to about 0.5 M of sodium or potassiumchloride is passed to the top of adsorbent bed R12 in an elution zone(R12-R10) to selectively desorb and recover the RuBisCO protein from theadsorbent in the elution zone and provide a product stream in line 212.The adsorbent beds R12, R11, and R10 are arranged in series, such thatthe R12 effluent stream withdrawn from the bottom of adsorbent bed R12in line 210 is passed to the top of adsorbent bed R11, and the R11effluent stream in line 211 is passed to the top of adsorbent bed R10. Aregeneration buffer stream in line 202 comprising sodium or potassiumhydroxide or a suitable base and sodium or potassium chloride is passedto the top of adsorbent bed R14 in a regeneration zone (R14-R13) toregenerate the adsorbent in the regeneration zone and provide a thirdwaste stream in line 209. The adsorbent beds R14 and R13 are arranged inseries, such that the R14 effluent stream withdrawn from the bottom ofadsorbent bed R12 in line 208 is passed to the top of adsorbent bed R13.A water stream in line 201 is passed to the top of adsorbent bed R15 anda fourth waste stream is withdrawn from the bottom of adsorbent bed R15.An equilibration buffer stream in line 206 having a concentration of 40to 100 mM of sodium or potassium phosphate and 0.1 M NaCl is passed tothe top of adsorbent bed R3 in the equilibration zone (R3-R1) toreionize and restore the adsorbent in the equilibration zone and providea fourth waste stream in line 221. The fourth waste stream in line 221may be neutralized and passed to waste disposal in a conventional mannerThe adsorbent beds R3, R2, and R1 are arranged in series, such that theR3 effluent stream withdrawn from the bottom of adsorbent bed R2 in line219 is passed to the top of adsorbent bed R2, and the R2 effluent streamin line 220 is passed to the top of adsorbent bed R1. The water washstream removes or flushes any residual salts from the adsorbent inadsorbent bed R15 to prevent any buildup of salts in the system prior tothe next feed loading cycle. At the completion of the cycle, theadsorbent beds are indexed by one adsorbent bed to the left, such thatthe last bed in the series R15 is moved to the first position and R14 ismoved to the water wash zone, or the last position. Using the SMBprocess with an adsorbent such as the strong basic exchange resin, suchas TOYOPEARL GIGACAP Q-650, the RuBisCO protein extraction yield wasabout 72 percent on a weight basis based on the amount of RuBisCOprotein in the feed, and the recovered RuBisCO protein purity was about85 wt-%.

In a still further embodiment of the invention is illustrated using 15adsorption beds in FIG. 10 for simulated moving bed (SMB) separationprocess for the continuous separation or extraction of multiple plantproteins, such as multiple proteins in pea protein, from pea flour. Thecrude feed mixture comprising the pea flour is diluted or dispersed inwater or an equilibration buffer stream comprising sodium or potassiumor sodium phosphate and sodium or potassium chloride in water. As shownin FIG. 10, the simulated moving bed comprises 15 adsorption beds(T1-T15) disposed in adsorption zones: Regeneration Zone (T15-T13),Elution Zone 3 (T12-T11), Elution Zone 2 (T10-T9), Elution Zone 1(T8-T7), Washing Zone (T6-T5), Feed Loading Zone (T4-T3), andEquilibration Zone (T2-T1). Each adsorbent bed has a top and a bottom.The individual adsorption beds (T1-T15) and are arranged serially fromleft to right, and grouped in the above six functionally isolatedsegments. T1 is in a first position and T15 is in the last position.Each isolated segment comprises at least one or more of the adsorbentbeds. In each isolated segment an external stream is introduced to thetop of a first adsorbent bed in the isolated segment and a waste streamor a product stream is withdrawn from the bottom of the last adsorbentbed (lower number adsorbent bed) in sequence, serially, from left toright. Each of the adsorbent beds contains a stationary phase adsorbentwhich is selective for the adsorption of protein of interest asdescribed hereinabove. According to FIG. 10, an SMB feed streamcomprising the crude protein in line 106 having a pea flourconcentration of about 1 wt-% is introduced to the top of adsorbent bedT4 in the feed loading zone (T4-T3). The adsorbent beds T4 and T3 arearranged in series, such that the T4 effluent stream withdrawn from thebottom of adsorbent bed T4 in line 119 is passed to the top of adsorbentbed T3. In the feed loading zone, the proteins of interest are disposedon the stationary phase adsorbent in adsorbent beds T4 and T3, and afirst waste stream is withdrawn from adsorbent bed T3 in line 120 andpassed to neutralization and waste disposal. A wash buffer stream inline 105 comprising sodium or potassium phosphate and sodium orpotassium chloride is passed to the top of adsorbent bed T6 in a washingzone (T6-T5) to wash impurities and other proteins from the adsorbent inthe washing zone and provide a second waste stream in line 118. Theadsorbent beds T6 and T5 are arranged in series, such that the T6effluent stream withdrawn from the bottom of adsorbent bed T6 in line117 is passed to the top of adsorbent bed T5. A first elution bufferstream in line 104 comprising sodium or potassium chloride is passed tothe top of adsorbent bed T8 in a first elution zone (T8-T7) toselectively desorb and recover a first group (Group A) of pea proteinsof interest from the adsorbent in the first elution zone (T8-T7) andprovide a first product stream in line 116. The adsorbent beds T8 and T7are arranged in series, such that the T8 effluent stream withdrawn fromthe bottom of adsorbent bed T8 in line 115 is passed to the top ofadsorbent bed T7. A second elution buffer stream in line 103 comprisingsodium or potassium chloride is passed to the top of adsorbent bed T10in a first elution zone (T10-T9) to selectively desorb and recover asecond group (Group B) of pea proteins of interest from the adsorbent inthe first elution zone (T10-T9) and provide a second product stream inline 114. The adsorbent beds T10 and T9 are arranged in series, suchthat the T10 effluent stream withdrawn from the bottom of adsorbent bedT10 in line 113 is passed to the top of adsorbent bed T9. A thirdelution buffer stream in line 102 comprising sodium or potassiumchloride is passed to the top of adsorbent bed T12 in a first elutionzone (T12-T11) to selectively desorb and recover a first group (Group C)of pea proteins of interest from the adsorbent in the third elution zone(T12-T11) and provide a first product stream in line 112. The adsorbentbeds T12 and T11 are arranged in series, such that the T12 effluentstream withdrawn from the bottom of adsorbent bed T12 in line 111 ispassed to the top of adsorbent bed T11. A regeneration buffer stream inline 101 comprising sodium or potassium chloride sodium and optionallypotassium hydroxide or a suitable base is passed to the top of adsorbentbed T15 in a regeneration zone (T15-T13) to regenerate the adsorbent inthe regeneration zone and provide a third waste stream in line 110. Theadsorbent beds T15, T14 and T13 are arranged in series, such that theT15 effluent stream withdrawn from the bottom of adsorbent bed T15 inline 108 is passed to the top of adsorbent bed T14, the T14 effluentstream withdrawn from the bottom of adsorbent bed T14 in line 109 ispassed to the top of adsorbent bed T13. An equilibration buffer streamin line 107 is passed to the top of adsorbent bed T2 in theequilibration zone (T2-T1). The adsorbent beds T1 and T2 are arranged inseries, such that the T1 effluent stream withdrawn from the bottom ofadsorbent bed T1 in line 121 is passed to the top of adsorbent bed T1. Afifth waste stream is withdrawn from adsorbent bed T1 in line 122 andpassed to neutralization and disposal. At the completion of the cycle,the adsorbent beds are indexed by one adsorbent bed to the left, suchthat the last bed in the series T15 is moved to the first position, andT14 is moved one position to the left, or the last position. Using theSMB process with an adsorbent such as the strong anion or strong basicexchange resin, such as TOYOPEARL GIGACAP Q-650, having a hydroxylatedpolymethacrylate polymer matrix.

A commercial embodiment of the SMB system of the current invention willarranged for maximum selectivity. The simulated moving bed operation isachieved by use of a plurality of adsorbent beds connected in series anda complex valve system, whereby the complex valve system facilitatesswitching at regular intervals the feed entry in one direction, themobile phase desorbent entry in the opposite direction, while changingthe eluted product and waste stream takeoff positions as well. The SMBsystem is a continuous process. Feed enters and the elute product(s) andwaste streams are withdrawn continuously at substantially constantcompositions. The overall operation is equivalent in performance to anoperation wherein the fluid and solid are contacted in a continuouscountercurrent manner, without the actual movement of the solid, orstationary phase adsorbent. In a commercial implementation of thepresent invention, the number of actual adsorbent beds in a particularzone of the SMB is a matter of economic choice and valve sizelimitations.

The following examples are provided to illustrate the present invention.These examples are shown for illustrative purposes, and any inventionembodied therein should not be limited thereto.

EXAMPLES Example 1 Quaternary Amine Cross-Linked Microcrystalline Resin

Part A: Cross-Linking of Microcrystalline Cellulose

The stationary phase adsorbent of the present invention is a quaternaryamine cross-linked microcrystalline resin. The quaternary aminecross-linked microcrystalline resin was prepared in the followingmanner. A 17.53 g of a general purpose emulsifier, RHODAFAC PE-510, apolyoxyethylenenonyl-phenyl ether phosphate (Available from SolvayChemicals, Houston, Tex.) was weighed in a 500 ml conical flask. A 400ml portion of cyclohexane was added to the conical flask, stirred for 30minutes, and allowed to rest over night at a room temperature of about25° C. A 65 g portion of AVICEL PH-200, a microcrystalline cellulose,(Available from FMC Corporation, Philadelphia, Pa.) having a particlesize ranging from about 150 to about 250 microns, an average particlesize of 180 um, a loose bulk density of 0.29-0.39 g/cc, and a moisturecontent of 2.0 to 5.0 wt-% was added to a two liter multi-neck reactionflask: a Morton Style round bottom flask, equipped with overheadstirrer, reflux condenser, thermocouple, and an addition funnel. Thereaction flask was placed in a heating mantle. A 52 ml portion of asolution of 45% sodium hydroxide and 250 ml of water was added to thereaction flask. The reaction mixture was vigorously stirred (370-380RPM) for 60 minutes while heating the reaction flask to maintain areaction temperature of 55° C. The heating was stopped, theemulsifier/cyclohexane mixture in the conical flask was added to thereaction mixture in the reaction flask. The reaction mixture was againstirred vigorously at a stirrer speed of about 420 rpm for an additional60 minutes while heating the reaction mixture to maintain a reactiontemperature of 55° C. A 75 ml first addition of epichlorohydrin wasadded to the reaction flask in a drop wise manner using the additionfunnel over a period of 20 minutes while stirring at 400 RPM andmaintaining the reaction flask at a reaction temperature of 55° C. Itwas observed at one point during the drop wise addition of the firstportion of epichlorohydrin that there was an exothermic reaction and abrief temperature excursion to about 59.1° C. Following the addition ofthe first portion of epichlorohydrin, the reaction mixture was againvigorously stirred at about 405 RPM, (revolutions per minute) foranother 60 minutes while maintaining the reaction mixture at about 55°C. At the end of this 60 minute period, a 45 ml (milliliter) portion ofa solution of 45 mol-% sodium hydroxide and 21 ml of water were added tothe reaction flask while stirring and maintaining the reaction flask at55° C. A second epichlorohydrin addition of 45 ml of epichlorohydrin wasadded to the reaction flask in a drop wise manner using the additionfunnel over a period of 10 minutes while stirring the reaction mixtureat a rate of 405 RPM and maintaining the reaction flask at a reactiontemperature of 55° C. Again, another temperature excursion occurredbriefly reaching 62.5° C. After complete addition of epichlorohydrin,the reaction mixture was stirred at 405 RPM while heating at 55° C. foran additional 60 minutes. The heating of the reaction flask was stoppedand the stirring at the rate of 405 RPM was continued for 2 hours. Thereaction mixture was then transferred to a 5 liter beaker containing 4liters of a 1 mol-% solution of sodium chloride in deionized water andallowed to rest for 30 minutes to permit the resulting gel to separatefrom a supernatant, having a cloudy appearance. The supernatant wasseparated from the gel by being decanted off and discarded. The 4 litersof a 1 mol-% solution of sodium chloride in deionized water was admixedwith the gel to wash the gel and the mixture of the sodium chloride andgel mixture was allowed to settle for 30 minutes to permit the gel tosettle out from the supernatant. The supernatant was again decanted offand discarded. This washing of the gel with the 1 mol-% sodium chloridesolution 4 more times to provide the washed cross-linked microcellulosegel. The washed cross-linked microcellulose gel was transferred to aBuchner funnel using SHARK SKIN filter paper, a creped, medium-to-slow,wet strength filter paper, having a 12 micron pore size and a diameterof 270 mm (Available from GE Healthcare Life Sciences, Marlborough,Mass.) and washed with 2 liters of 1 mol-% sodium chloride aqueoussolution. The resulting cross-linked microcellulose gel was stored in anaqueous solution of 1 mol-% sodium chloride.

Part B: Reaction of Cross-Linked Microcellulose with Quaternary Amine

QUAB 151 (Available from SKW QUAB Chemicals, Inc. Saddle Brook, N.J.) isthe an aqueous solution of the active substance2,3-epoxypropyltrimethyl-ammonium chloride (glycidyltrimethylammoniumchloride). The reaction of cross-linked microcellulose gel with thequaternary amine, QUAB 151 was carried out in a second 500 ml reactionflask. A 75 g portion of suction dried cross-linked microcellulosegelprepared in Part A of Example 1 was added to 50 ml of 0.6 N NaOHsolution containing sodium borohydride (2 mg/ml) in the second 500 mlreaction flask and stirred. 50 ml of the QUAB 151 was added to thesecond 500 ml reaction flask and stirred for 20 hours at roomtemperature (about 25° C.). The resulting reaction mixture was filteredin a Buchner funnel using a SHARK SKIN filter paper, a creped, medium toslow, wet strength filter paper, having a 12 micron pore size and adiameter of 110 mm (Available from GE Healthcare Life Sciences,Marlborough, Mass.) and washed with 3 liters of 1 mol-% sodium chlorideaqueous solution. The resulting filter cake, or quaternary aminecross-linked microcellulose resin was suction dried and stored at astorage temperature of 4° C.

Example 2 Recombinant Yeast Heme Protein Purification Using QuaternaryAmine Cross-Linked Microcellulose Resin

Approximately 5 gm (5 ml) sample of the adsorbent, the quaternary aminecross-linked microcelluloseresin prepared in Example 1, was packed in aplastic column having an inside diameter of 12 mm and a length of 45 mm.The quaternary amine cross-linked microcellulose resin was firstequilibrated by passing 5 column volumes of an equilibration buffer 1comprising a potassium phosphate solution having a potassium phosphateconcentration of 50 mM (millimolar) sodium phosphate, and having a pH8.0. The equilibration buffer was passed concurrently to the top of theglass column at a rate of 5 ml/min. The resin was then equilibrated bypassing 20 column volumes of an equilibration buffer 2 comprising apotassium phosphate solution having a potassium phosphate concentrationof 4 mM potassium phosphate, and having a pH 8.0. The effluent from thebottom of the column during the equilibration step was passed to wastedisposal. Following equilibration, a feed comprising yeast lysate wasloaded on the adsorbent in a feed loading step. A 38 mg sample of yeastlysate which had been stored at −20° C., was thawed at room temperatureand diluted with equilibration buffer to adjust the total proteinconcentration to ˜2 mg/ml at 4° C. to provide a feed mixture, and the pHof the feed mixture was adjusted to a pH of 8 with 1N NaOH on ice, andthe conductivity of the feed mixture was about 800 μS. The feed mixturehad protein concentration of 2.18 mg/ml which was determined by Pierce660 nm protein assay. The Pierce 660 nm Protein Assay (Available fromThermo Fischer Scientific, Pittsburgh, Pa.) uses a proprietary dye-metalcomplex which binds to protein in acidic conditions, causing a shift inthe dye's absorption maximum, which is measured at 660 nm. A 17.5 mlportion of the feed mixture was passed to the top of the column at afeed rate of about 0.5 ml/min and the resulting elute was collected 5 mlfractions. The column was then washed with 5 column volumes of a washbuffer comprising an aqueous solution having a potassium phosphateconcentration of 4 mM sodium phosphate, and a pH 8.0. The wash bufferwas passed to the top of the column at a wash rate of about 0.5 ml/minand the resulting wash effluent was collected in 5 ml fractions. In anelution step, the heme protein was then eluted by passing an elutionbuffer comprising potassium phosphate and sodium chloride to the top ofthe column and collecting the eluted heme protein product. The elutionbuffer comprised a potassium phosphate concentration of 4 mM, and aconcentration of sodium chloride of 25 mM sodium chloride and had a pHof 8.0. The elution buffer was passed to the top of the column at anelution rate of 0.5 ml/min (See FIG. 3), and the extracted heme proteinproduct was collected from the bottom of the column. The column was thenwashed with 5 column volumes of 1M NaCl solution at a rate of 2 ml/minto elute the other proteins. Protein concentration was again determinedby using Pierce 660 nm protein assay. Purity was determined by analyzingthe collected product fractions on 4-12% Bis-Tris SDS-PAGE followed bydensitometry using Bio-Rad's EZ Imager (See FIG. 4). NuPAGE Bis-Trisgels (Available from Thermo Fischer Scientific, Pittsburgh, Pa.) areprecast polyacrylamide gels designed to provide separation of small tomedium-sized proteins during gel electrophoresis. Bio-Rad's EZ IMAGER isan automated gel imaging instrument (Available from Bio-Rad, Hercules,Calif.). The adsorbent was regenerated in a regeneration step by washingthe column with 10 column volumes of 0.1 M NaOH+1 M NaCl followed bywashing the column with 5 column volumes of 0.1 N phosphoric acid. Thecolumn was then equilibrated in an equilibration step bypassing 5 columnvolumes at a rate of 5 ml/min of an equilibration buffer 1 comprising 50mM potassium phosphate, and having a pH of 8.0. The resin was thenequilibrated by passing 20 column volumes of an equilibration buffer 2comprising a potassium phosphate solution having a potassium phosphateconcentration of 4 mM potassium phosphate, and having a pH 8.0. Theprotein of interest was leghemoglobin protein, and the lysate wasdetermined to contain about 25% percent leghemoglobin protein by weight.The leghemoglobin protein content of the lysate was determined bydensitometry to contain 9.53 mg of the approximately 38 mg loaded. Theleghemoglobin protein found in the protein product elute fractions(shown in FIG. 4 as Lanes 5-9) was determined to be 11.16 mg. Thus, theheme protein extraction yield was about 97% on a weight basis comparedto the amount of heme protein in the feed, and the heme protein puritywas about 83%, determined by densitometry.

The analytical results for the extraction of heme protein from thelysate of Example 2 were depicted in graphical form in FIG. 3 and FIG.4.

FIG. 3 shows the optical density area plot of the protein concentrationof the numbered elute fractions withdrawn from the column in Example 2.

FIG. 4 is gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced in Example 2.FIG. 4 shows the collected elute fractions eluted from the column ofExample 2 containing the quaternary amine cross-linked microcelluloseresin on 4-12% Bis-Tris SDS-PAGE imager at various points in the SMBprocess. The results are shown in lanes which are numbered along thex-axis. Lane1: Feed load, 14 ug (micrograms); Lane 2: Elution pool, 1.08ug; Lane 3: Elution pool, 1.62 ug; Lane 4: Elution pool, 2.16 ug; Lanes5: Elution pool, 2.7 ug; Lane 6: 3.1 ug, Lane 7: Molecular weight markerproteins.

Example 3 Heme Protein Purification Using Chromatography with StrongAnion Exchange Resin in a Single Column Separation

Example 3 evaluated DIAION HPA25L a strongly basic anion exchange resinhaving a backbone matrix of styrene divinyl benzene polymer (Availablefrom Mitsubishi Chemical Company, Tokyo, Japan) for use in a singlecolumn separation process to determine the ability of the HPA25L resinto provide the required degree of separation and also be regenerableduring an SMB cycle.

A. Resin Preparation:

Accordingly, a resin volume of about 5 ml of DIAION HPA25L resin waswashed in a resin wash step to remove impurities and any fines. Theresin wash step consisted of soaking the volume of the resin in twotimes the volume (about 10 ml) of resin in a 1:1 (v/v) mixture of 1 MNaOH: methanol for 2 hours at room temperature and at atmosphericpressure. At the end of 2 hours, the supernatant was decanted and theremaining resin solids were washed again with in two times the volume ofresin in a 1:1 (v/v) mixture of 1 M NaOH: methanol for 2 hours at roomtemperature and at atmospheric pressure. After decanting thesupernatant, the resin was washed three times with water by soaking thevolume of resin in twice the volume of water and allowing theresin/water mixture to stand for about 30 minutes, before decanting thewater. The resin was then washed 3 times with a 2 N HCl solution bysoaking the resin in twice the volume of HCl solution and allowing theresin/HCl solution to stand for 30 minutes and then decanting the HClsolution. The washed resin was packed in a high-modulus polyethylene,(HMPE) column (having a diameter of about 12 mm and a length of about 45mm) and washed with water until the eluate had a pH of about 4. Theresin was then washed with 3 column volumes of 20 vl-% ethanol andstored at room temperature.

B. Heme (leghemoglobin) Protein Purification:

The washed resin (approximately 5 g or 5 ml) in the packed HMPE column,prepared hereinabove in Example 3—Part A, was washed with 5 columnvolumes of 50 mM (milliMolar) sodium phosphate solution having a pH of8.0 at a rate of 0.5 ml/min. The resin was equilibrated by passing 10column volumes of a 5 mM sodium phosphate solution having a pH of 8.0 at0.5 ml/min. A 34.125 mg portion of yeast lysate which had been stored ata storage temperature of −20° C. was diluted a 5 mM sodium phosphatesolution having a pH of 8.0 to provide about a 17.5 ml feed mixturewhich was maintained on ice at a temperature of about 4° C. The feedmixture had a conductivity of about 1.0 mS (milliSiemans). In a loadingstep, the 17.5 ml of the feed mixture was passed through the column orloaded on the resin at a rate of about 5 ml/min and the eluate wascollected in 5 ml fractions. In a washing step, the column was washedwith about 5 column volumes of a 5 mM solution of sodium phosphatehaving a pH of 8.0 at a rate of 0.5 ml/min and the resulting wash eluatewas collected in 5 ml fractions. The heme protein remaining on the resinafter the wash step was eluted in an elution step by flushing the columnwith 5 column volumes of an elution buffer of 5 mM sodium phosphate and50 mM sodium chloride and having a pH of 8.0. The protein eluate wascollected at 5 ml fractions and stored on ice at a temperature of 4° C.The remaining proteins on the resin were eluted by flushing the columnwith a 1 M solution of sodium chloride. The protein concentration in thecollected fractions was determined by Pierce 660 nm protein assay. ThePierce 660 nm Protein Assay (Available from Thermo Fischer Scientific,Pittsburgh, Pa.) uses a proprietary dye-metal complex which binds toprotein in acidic conditions, causing a shift in the dye's absorptionmaximum, which is measured at 660 nm. Purity of the protein wasdetermined by analyzing the collected product fractions on 4-12%Bis-Tris SDS-PAGE followed by densitometry using Bio-Rad's EZ Imager(See FIG. 6). NuPAGE Bis-Tris gels (Available from Thermo FischerScientific, Pittsburgh, Pa.) are precast polyacrylamide gels designed toprovide separation of small to medium-sized proteins during gelelectrophoresis. Bio-Rad's EZ IMAGER is an automated gel imaginginstrument (Available from Bio-Rad, Hercules, Calif.). The heme proteincontent of the lysate was determined by densitometry to contain 7.47 mgof the approximately 34.125 mg loaded. The heme protein found in theprotein product elute fractions (shown in FIG. 6 as Lanes 7-10) wasdetermined to be 5.9 mg. Thus, using the strong basic resin DIAIONHPA25L the heme protein extraction yield was about 79% on a weight basiscompared to the amount of heme protein in the feed, and the heme proteinpurity was about 79%, determined by densitometry.

FIG. 5 shows the optical density area plot of the protein concentrationsof the numbered elute fractions withdrawn from the column in Example 3.

FIG. 6 is gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced in Example 3.FIG. 6 shows the collected elute fractions eluted from the column ofExample 3 on 4-12% Bis-Tris SDS-PAGE imager at various points in the SMBprocess. The results are shown in lanes which are numbered along thex-axis. Lane 1: Lysate 5 ul (microliters); Lane 2: Lysate 6 ul; Lane 3:Lysate 8 ul; Lane 4: Lysate 10 ul; Lane 5: FT 30 ul; Lane 6: Wash 30 ul;Lane 7: Elution pool 10 ul; Lane 8: Elution pool 15 ul; Lane 9: Elutionpool 20 ul, 2.21 ug (micrograms) heme protein of interest, and Lane 10:Elute pool 25 ul.

Example 4 Plant (RuBisCO) Protein Purification Using Single ColumnPurification with Strong Anion Exchange Resin

Example 4 represented a single column purification of the plant protein,RuBisCO (1,5-bisphosphate carboxylase/oxygenase) protein from spinachextract. The stationary phase employed in the single column wasTOYOPEARL GIGACAP Q-650, a high capacity, high resolution, strong anionexchange resin having a backbone of a hydroxylated polymethacrylatepolymer (Available from Tosoh Bioscience LLC, King of Prussia, Pa.). A 5ml portion (column volume) of the resin was loaded in a column having adiameter of about 12 mm and a length of about 45 mm. The resin wasequilibrated by passing 10 column volumes of an equilibration buffercomprising a 40 mM sodium phosphate and 0.1 M sodium chloride solutionhaving a pH of 8.0 at 0.5 ml/min. A 0.5 g/liter crude feed mixture ofspinach extract was prepared by admixing the spinach extract in theequilibration buffer. In a loading step, the 2.8 liters of the crudefeed mixture was passed through the column or loaded on the resin at arate of about 10 ml/min. In a washing step, the column was washed withabout 40 column volumes of a 40 mM solution of sodium phosphate and 0.15M sodium chloride having a pH of 8.0 at a rate of 10 ml/min. The proteinof interest, RuBisCO, remaining on the resin after the wash step waseluted in an elution step by flushing the column with 20 column volumesof an elution buffer having an elution buffer concentration of 40 mMsodium phosphate and 0.35 M sodium chloride and having a pH of 8.0, andintroduced at a rate of 10 ml/min. The protein eluate was collected at 5ml fractions and stored on ice at a temperature of 4° C. The RuBisCOprotein concentration in the collected fractions was determined byPierce 660 nm protein assay. The Pierce 660 NM Protein Assay (Availablefrom Thermo Fischer Scientific, Pittsburgh, Pa.) uses a proprietarydye-metal complex which binds to protein in acidic conditions, causing ashift in the dye's absorption maximum, which is measured at 280 nm.Purity of the protein was determined by analyzing the collected productfractions on 4-12% Bis-Tris SDS-PAGE followed by densitometry usingBio-Rad's EZ Imager (See FIG. 6). NuPAGE Bis-Tris gels (Available fromThermo Fischer Scientific, Pittsburgh, Pa.) are precast polyacrylamidegels designed to provide separation of small to medium-sized proteinsduring gel electrophoresis. Bio-Rad's EZ IMAGER is an automated gelimaging instrument (Available from Bio-Rad, Hercules, Calif.). TheRuBisCO protein content of the spinach extract was determined bydensitometry to contain 7.47 mg of the approximately 34.125 mg loaded.The RuBisCO protein found in the protein product elute fractions (shownin FIG. 6 as Lanes 7-10) was determined to be 5.9 mg. Thus, using thestrong basic resin TOYOP EARL GIGACAP Q-650, the RuBisCO proteinextraction yield was about 72 percent on a weight basis compared to theamount of RuBisCO protein in the feed, and the RuBisCO protein puritywas about 85 wt-%, determined by densitometry.

FIG. 7 shows the elution profile of RuBisCO from spinach in an opticaldensity area plot of the protein concentrations of the numbered elutefractions withdrawn from the column in Example 4.

FIG. 8 is gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced in Example 4.The results are shown in lanes which are numbered along the x-axis.Lanes 1-7: Purified RuBisCO protein and Lane 8: Molecular weight markerproteins.

Example 5 Pea Protein Purification Using a Single Column Purificationwith Strong Anion Exchange Resin

Example 5 represented a Single Column purification of Pea protein frompea flour. The stationary phase employed in the SMB was TOYOPEARLGIGACAP Q-650, a high capacity, high resolution, strong anion exchangeresin having a backbone of a hydroxylated polymethacrylate polymer(Available from Tosoh Bioscience LLC, King of Prussia, Pa.). A 5 mlportion (column volume) of the resin was loaded in a column having adiameter of about 12 mm and a length of about 45 mm. The resin wasequilibrated by passing 20 column volumes of an equilibration buffercomprising a 20 mM sodium phosphate and 0.05 M sodium chloride solutionhaving a pH of 8.0 at 2 ml/min. A 1 wt-% pea flour SMB feed mixture ofthe pea flour was prepared by admixing about 1 g/100 ml of the pea flourin the equilibration buffer. The total protein concentration in thecrude feed mixture was 5.2 mg/ml as determined by Pierce 660 NM proteinassay. In a loading step, the 50 ml of the crude feed mixture was passedthrough the column or loaded on the resin at a rate of about 2 ml/min.In a washing step, the column was washed with about 5 column volumes ofa 20 mM sodium phosphate and 0.05 M sodium chloride solution having a pHof 8.0 at a rate of 2 ml/min. The proteins of interest, 3 targetproteins (Group A, B, and C), remaining on the resin after the wash stepwere eluted in an elution step by flushing the column with 4 columnvolumes of an elution buffer having a linear gradient of from 50 mM to500 mM sodium chloride in 20 mM sodium phosphate and having a pH of 8.0at a rate of 2 ml/min. The protein eluate was collected at 5 mlfractions and stored on ice at a temperature of 4° C. The pea proteinconcentrations in the collected fractions were determined by Pierce 660nm protein assay at 280 nm. Purity of the protein was determined byanalyzing the collected product fractions on 4-12% Bis-Tris SDS-PAGEfollowed by densitometry using Bio-Rad's EZ Imager (See FIG. 6). NuPAGEBis-Tris gels (Available from Thermo Fischer Scientific, Pittsburgh,Pa.) are precast polyacrylamide gels designed to provide separation ofsmall to medium-sized proteins during gel electrophoresis. There were 3target proteins identified as Target A, Target B, and Target C, Thetotal protein found was as follows: Target group A proteins −51 mg,Target group B proteins −90 mg, and Target group C proteins 5 mg. Theproteins in the eluted fractions was determined using a Pierce 660 NMprotein assay.

FIG. 11 shows the elution profile of pea proteins extracted from peaflour in an optical density area plot (UV Absorbance @ 280 nm) of thepea protein concentrations of the numbered elute fractions withdrawnfrom the column in Example 5.

FIG. 12 is gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced in Example 5.The results are shown in lanes which are numbered along the x-axis.Lanes 2-4 contain Target group A pea proteins, Lanes 5-7 contain Targetgroup B pea proteins, and Lanes 15-16 contain Target group C peaproteins. Lane 17 shows molecular weight marker proteins.

Example 6 Simulated Moving Bed Purification of Heme Protein in an 8-BedSimulated Moving Bed Separation

Approximately 20 ml of DIAION HPA25L a strongly basic anion exchangeresin having a backbone matrix of styrene divinyl benzene polymer(Available from Mitsubishi Chemical Company, Tokyo, Japan) was loaded ineach of eight 20 ml cartridges. Each cartridge was a vertical columnhaving a diameter of 21 mm and a height of 80 mm. Each of the cartridgeswere prepared by passing about 30 column volumes of an equilibrationbuffer comprising 5 mM of potassium phosphate at a pH of 8.0 at a rateof 20 ml/min. The yeast lysate to be purified was kept frozen. A 200 mlportion of the frozen yeast lysate was thawed at room temperature (25°C.) and diluted to a volume of 650 ml by the addition of theequilibration buffer to provide a crude feed mixture. The crude feedmixture pH was adjusted to 8.0 by the addition of a sufficient amount ofa 1N solution of sodium hydroxide. The crude feed was maintained at afeed temperature of about 4° C. with ice. The conductivity of the crudefeed mixture was about 0.9 mS/cm. A bench scale OCTAVE simulated movingbed unit (Available from Semba Biosciences, Inc., Madison, Wis.) wasconfigured with eight beds according to the process flow of FIG. 13. TheSMB process took place at room temperature and atmospheric pressure, butthe feed mixture and the eluted product streams were maintained at atemperature of about 4° C. on ice. According to FIG. 13, the crude feedmixture was introduced in line 300 to load adsorbent bed O1 (loadingzone) and a first waste stream is withdrawn in line 350. A wash bufferis introduced to adsorbent bed O2 (wash zone) in line 302 and a secondwash stream is withdrawn from adsorbent bed O2 in line 340. An elutionbuffer in line 304 is introduced to adsorbent bed O5 which is in serialfluid communication with adsorbent beds O4 and O3 (elution zone O5-O3)and a product stream is withdrawn from adsorbent bed O3 in line 300.Adsorbent beds O7 and O6 (regeneration zone) are counter currentlyregenerated in line 306 by passing a first regeneration buffer to thebottom of regeneration bed O7 which is in serial fluid communicationwith adsorbent bed O6 and a third waste stream is withdrawn fromadsorbent bed O6 in line 320. A second regeneration buffer and a firstequilibration buffer are also introduced in line 306 to complete theregeneration process and initiate the equilibration process at theappropriate point in the SMB cycle. The second equilibration buffer isintroduced in line 308 to adsorbent bed O8 (equilibration zone) and afourth waste stream is withdrawn in line 310. The crude feed rate was 2ml/ml. The wash buffer comprised 5 column volumes of 5 mM potassiumphosphate at a pH of 8.0 and was introduced at a rate of 2 ml/min. Theelution step comprised passing an elution buffer of 10 column volumes of5 mM potassium phosphate and 50 mM sodium chloride at a rate of 2ml/min. The regeneration step comprised passing 5 column volumes of afirst regeneration buffer of 2 M sodium hydroxide and 1 M sodiumchloride and a rate of 1.66 ml/min, followed by passing a secondregeneration buffer of 5 column volumes of 1% HCl at 20 ml/min, followedby passing a first equilibration buffer of 5 column volumes of 50 mMpotassium phosphate having a pH of 8.0 at a rate of 20 ml/min. Thesecond equilibration buffer of 30 column volumes of 5 mM potassiumphosphate and having a pH of 8.0 was passed to O8 in line 308 at a rateof 20 ml/min. The elute product stream in line 330 was monitored at a UVwave length of 280 NM and the protein concentration was determined by aPierce 660 NM protein assay. The purity of the elute product wasdetermined by analyzing the eluted fractions on a 4-12% Bis-TrisSDS-Page followed by densitometry using a Bio-Rad's Gel Doc EZ Imager asdescribed hereinabove. The total amount of protein in the crude feedmixture was 1120 mg. The protein of interest, leghemoglobin, wasdetermined to be 26 wt-% by densitometry and equal to 291 mg. The SMBproduct eluted from the SMB was 301.6 mg and the purity of the SMBproduct was determined by densitometry to be 80 wt-%. Thus, the amountof pure (100%) leghemoglobin in the product was 241 mg, and the overallyield of the leghemoglobin was 83 wt-%, based on the amount ofleghemoglobin in the crude feed mixture.

While the disclosure has been described in terms of specificembodiments, it is evident in view of the foregoing description thatnumerous alternatives, modifications and variations will be apparent tothose skilled in the art. Accordingly, the disclosure is intended toencompass all such alternatives, modifications and variations which fallwithin the scope and spirit of the disclosure and the following claims.

FIG. 14 shows the optical density of the elute fractions withdrawn fromthe Simulated Moving Bed process in Example 6.

FIG. 15 is gel electrophoresis result of an SDS-PAGE protein analysisusing 4-12% Bis-Tris Gel of the elute fractions produced in Example 6.FIG. 14 shows the collected elute fractions eluted from the column ofExample 6 on 4-12% Bis-Tris SDS-PAGE imager at various points in the SMBprocess. The results are shown in lanes which are numbered along thex-axis. Lane 1: Lysate 5 μl; Lane 2: Lysate 10 ul, Lane 3: Flowthrough30 ul; Lane 4: Wash 30 ul; Lane 5: Elute Pool 10 ul; Lane 6: Elute Pool:25 μl; Lane 7: Elute Pool 15 ul; Lane 8: Molecular weight markerProteins.

We claim:
 1. A process for the continuous extraction of at least oneprotein of interest from a crude protein in a continuous simulatedmoving bed (SMB) extraction zone, said process comprising: a) admixing acrude protein comprising the at least one protein of interest selectedfrom the group consisting of a heme protein, RuBisCO, and a pea protein,other proteins, and impurities with an equilibration buffer streamcomprising a phosphate salt of sodium or potassium and water to providean SMB feed stream: b) continuously passing the SMB feed stream at SMBfeed conditions and a plurality of wash streams to the continuoussimulated moving bed (SMB) extraction zone, the continuous SMBextraction zone having a plurality of adsorbent beds, one or more of theadsorbent beds being disposed in a loading zone, a washing zone, aelution zone, a regeneration zone, an equilibration zone, and optionallya capture zone or a second washing zone, each of said zones beingsequentially disposed in parallel from the loading zone having a firstadsorbent bed in a first position to the equilibration zone having alast adsorbent bed in a last position, each of the loading zone, thewashing zone, the elution zone, the equilibration zone, and theequilibrium zone containing the one or more adsorbent beds, each of theone or more adsorbent beds having a top and a bottom and each of the oneor more adsorbent beds containing an adsorbent selected from the groupconsisting of a strong basic anion exchange resin having a backbonematrix comprising a cross linked styrene divinyl benzene polymer, astrong anion exchange resin having a backbone of a hydroxylatedpolymethacrylate polymer and a quaternary amine cross linkedmicrocrystalline resin comprising a microcrystalline cellulose which hasbeen cross linked with epichlorohydrin and reacted with2,3-epoxypropyltrimethyl-ammonium chloride, said plurality of washstreams having a pH of from about 7.5 to about 8.5 including a washbuffer stream, an elution buffer stream, a regeneration buffer stream, asecond regeneration buffer stream, and the equilibration buffer stream,said continuous SMB extraction zone comprising: i) concurrently passingthe SMB feed stream at SMB feed conditions to the top of the loadingzone containing a first adsorbent bed to load the SMB feed mixture onthe adsorbent in the loading zone and withdrawing a first waste streamcomprising water and impurities from the bottom of the loading zone; ii)concurrently passing the wash buffer stream comprising phosphate salt ofsodium or potassium to the top of the washing zone to wash the adsorbentin the wash zone to provide a wash effluent stream and withdrawing thewash effluent stream from the bottom of the wash zone; iii) concurrentlypassing the elution buffer comprising phosphate salt of sodium orpotassium and chloride salt of sodium or potassium to the top of theelution zone and eluting an elution effluent stream comprising theprotein of interest from the bottom of the elution zone; iv)concurrently or counter-currently passing the regeneration buffercomprising phosphate salt of sodium or potassium and a base to theregeneration zone, and concurrently or counter-currently passing thesecond regeneration buffer stream comprising an acid to the regenerationzone, and withdrawing a second waste stream from the regeneration zone;v) concurrently passing the equilibration buffer comprising phosphatesalt of sodium or potassium and optionally a chloride salt of sodium orpotassium to the top of the equilibration zone comprising the lastadsorbent bed to restore ionic activity to the adsorbent in theequilibration zone and withdrawing a third waste stream from the bottomof the equilibration zone; and, vi) indexing the adsorbent beds whereinthe first adsorbent bed in the loading zone is advanced by one adsorbentbed toward the equilibration zone and the last adsorbent bed in theequilibration zone is repositioned to the first position in the loadingzone to provide the continuous SMB extraction zone; vii) passing thefirst waste stream, the second waste stream, the third waste stream, andat least a portion of the wash effluent stream to waste disposal; and,viii) recovering the elution effluent stream as a protein product streamcomprising the protein of interest selected from the group consisting ofa heme protein, RuBisCO, and a pea protein.
 2. The process of claim 1,wherein the SMB feed conditions include an SMB feed temperature of fromabout 4° C. to about 25° C.
 3. The process of claim 1, wherein theequilibration buffer stream comprises an equilibration bufferconcentration of from about 2 mM to about 100 mM of phosphate salt ofsodium or potassium, and optionally from about 0.05M to about 0.1M ofsodium or potassium chloride.
 4. The process of claim 1, wherein theelution buffer stream comprises an elution buffer concentration of fromabout 2 mM to about 100 mM of phosphate salt of sodium or potassium, andfrom about 0.02 M to about 0.5 M of sodium or potassium chloride.
 5. Theprocess of claim 1, wherein the wash buffer stream comprises a washbuffer concentration of from about 2 mM to about 100 mM of phosphatesalt of sodium or potassium.
 6. The process of claim 1, wherein theregeneration buffer stream comprises a regeneration buffer concentrationof from about 0.1 to about 2 M of sodium or potassium hydroxide andabout 0.5-2 M of sodium or potassium chloride.
 7. The process of claim1, wherein the SMB elution effluent stream is maintained at a pH ofabout 7.5 to 8.5 and a collection temperature of about 4° C. or less. 8.The process of claim 1, wherein the crude protein comprises a plantprotein comprising the protein of interest being RuBisCO and thecontinuous SMB extraction zone further comprises the second washing zonedisposed between the regeneration zone and the equilibration zone andpassing a second wash buffer comprising water to the second washing zoneand withdrawing a second washing zone effluent from the second washingzone and admixing the second washing zone effluent with the second wastestream.
 9. The process of claim 1, wherein the crude protein comprises afirst pea protein of interest, a second pea protein of interest, and athird pea protein of interest and the continuous SMB extraction zonefurther comprises a second elution zone, and a third elution zone andpassing a second elution buffer to the second elution zone to recover asecond pea protein product stream, passing a third elution buffer to thethird elution zone to recover a third pea protein product stream,wherein the elution buffer, the second elution buffer, and the thirdelution buffer each contain an effective amount of sodium or potassiumsalt to displace the first, second and third protein of interest. 10.The process of claim 1, wherein the effective amount of sodium orpotassium salt to displace the first pea protein of interest comprisesfrom about 0.05 to about 0.2 M potassium or sodium phosphate, whereinthe effective amount of sodium or potassium salt to displace the secondpea protein of interest comprises from about 0.2 to about 0.35 Mpotassium or sodium phosphate, and wherein the effective amount ofsodium or potassium salt to displace the third pea protein of interestcomprises from about 0.35 to about 0.5 M potassium or sodium phosphate.11. The process of claim 1, wherein the quaternary amine cross linkedmicrocrystalline resin comprises a particle size ranging from about 150to about 250 microns, and a loose bulk density of from about 0.29 toabout 0.39 g/cc and being cross-linked with epichlorohydrin andexchanged with 2,3-epoxypropyltrimethyl-ammonium chloride.
 12. Theprocess of claim 1, wherein SMB feed conditions include a feedconcentration of about 1 to about 10 mg/ml, an SMB feed temperatureabout 4° C. and an SMB feed pH of between about 7.5 and about 8.5. 13.The process of claim 1, wherein the wash buffer stream comprisespotassium phosphate and has a pH of about 7.5 to 8.5 and a conductivityof between 0.5 and 25 mS/cm.
 14. The process of claim 1, wherein theprotein of interest is a heme protein and the continuous SMB extractionzone further comprises: a) the capture zone disposed before the loadingzone and after the equilibration zone and passing at least a portion ofthe wash effluent stream to the top of the capture zone and withdrawinga capture zone effluent from the bottom of the capture zone to minimizelosses of the protein of interest in the wash zone and admixing thecapture zone effluent with the first waste stream; b) a secondregeneration zone disposed between the regeneration zone and theequilibration zone and counter currently or concurrently passing thesecond regeneration buffer stream comprising HCl to the secondregeneration zone and withdrawing a second regeneration zone effluentstream and admixing the second regeneration effluent stream with thesecond waste stream; and, c) a second equilibration zone disposedbetween the second regeneration zone and the capture zone and passing asecond equilibration buffer stream comprising sodium or potassiumphosphate to the top of the second regeneration zone, withdrawing asecond regeneration zone effluent and admixing the second regenerationzone effluent with the third waste stream.
 15. The process of claim 14,wherein the wash buffer comprises a potassium phosphate concentration offrom about 2 to about 10 mM.
 16. The process of claim 14, wherein theelution buffer stream comprises potassium phosphate and sodium chlorideand having a pH of about 7.8 to about 8.2 and a conductivity of fromabout 0.5 to about 10 mS/cm.
 17. The process of claim 14, whereinregeneration buffer stream comprises about 0.1 N to about 2 N NaOHsodium hydroxide and about 0.5 to about 2M sodium chloride.
 18. Theprocess of claim 14, wherein the second regeneration buffer streamcomprises from about 0.05 to about 0.3 N HCl or H₃PO₄.
 19. The processof claim 14, wherein the equilibration buffer stream comprises from 2 toabout 10 mM potassium phosphate and having a pH of about
 8. 20. Theprocess of claim 14, wherein the second equilibration buffer streamcomprises from about 2 to about 10 mM potassium phosphate and having apH of about
 8. 21. The process of claim 1, wherein the adsorbent is astrong basic anion exchange resin having a backbone matrix comprising across linked styrene divinyl benzene polymer.
 22. The process of claim1, wherein the adsorbent is a quaternary amine cross linkedmicrocrystalline resin comprising a microcrystalline cellulose which hasbeen cross linked with epichlorohydrin and reacted with2,3-epoxypropyltrimethyl-ammonium chloride.
 23. A process for thecontinuous extraction of at least one protein of interest from a crudeprotein in a continuous simulated moving bed (SMB) extraction zone, saidprocess comprising: c) admixing a crude protein comprising the at leastone protein of interest selected from the group consisting of a hemeprotein, RuBisCO, and a pea protein, other proteins, and impurities withan equilibration buffer stream comprising a phosphate salt of sodium orpotassium and water to provide an SMB feed stream: d) continuouslypassing the SMB feed stream at SMB feed conditions and a plurality ofwash streams to the continuous simulated moving bed (SMB) extractionzone, the continuous SMB extraction zone having a plurality of adsorbentbeds, one or more of the adsorbent beds being disposed in a loadingzone, a washing zone, a elution zone, a regeneration zone, anequilibration zone, and optionally a capture zone or a second washingzone, each of said zones being sequentially disposed in parallel fromthe loading zone having a first adsorbent bed in a first position to theequilibration zone having a last adsorbent bed in a last position, eachof the loading zone, the washing zone, the elution zone, theequilibration zone, and the equilibrium zone containing the one or moreadsorbent beds, each of the one or more adsorbent beds having a top anda bottom and each of the one or more adsorbent beds containing anadsorbent comprising a quaternary amine cross linked microcrystallineresin comprising a microcrystalline cellulose which has been crosslinked with epichlorohydrin and reacted with2,3-epoxypropyltrimethyl-ammonium chloride and having a particle size offrom about 150 to 250 microns, said plurality of wash streams having apH of from about 7.5 to about 8.5 including a wash buffer stream, anelution buffer stream, a first regeneration buffer stream, a secondregeneration buffer stream, and the equilibration buffer stream, saidcontinuous SMB extraction zone comprising: i) concurrently passing theSMB feed stream at SMB feed conditions to the top of the loading zonecontaining a first adsorbent bed to load the SMB feed mixture on theadsorbent in the loading zone and withdrawing a first waste streamcomprising water and impurities from the bottom of the loading zone; ii)concurrently passing the wash buffer stream comprising phosphate salt ofsodium or potassium to the top of the washing zone to wash the adsorbentin the wash zone to provide a wash effluent stream and withdrawing thewash effluent stream from the bottom of the wash zone; iii) concurrentlypassing the elution buffer comprising phosphate salt of sodium orpotassium and chloride salt of sodium or potassium to the top of theelution zone and eluting an elution effluent stream comprising theprotein of interest from the bottom of the elution zone; iv)concurrently or counter-currently passing the first regeneration buffercomprising phosphate salt of sodium or potassium and a base to theregeneration zone, concurrently or counter-currently passing the secondregeneration buffer comprising HCl or H₃PO₃, and withdrawing a secondwaste stream from the regeneration zone; v) concurrently passing theequilibration buffer comprising phosphate salt of sodium or potassiumand optionally a chloride salt of sodium or potassium to the top of theequilibration zone comprising the last adsorbent bed to restore ionicactivity to the adsorbent in the equilibration zone and withdrawing athird waste stream from the bottom of the equilibration zone; and, vi)indexing the adsorbent beds wherein the first adsorbent bed in theloading zone is advanced by one adsorbent bed toward the equilibrationzone and the last adsorbent bed in the equilibration zone isrepositioned to the first position in the loading zone to provide thecontinuous SMB extraction zone; vii) passing the first waste stream, thesecond waste stream, the third waste stream, and at least a portion ofthe wash effluent stream to waste disposal; and, viii) recovering theelution effluent stream as a protein product stream comprising theprotein of interest selected from the group consisting of a hemeprotein, RuBisCO, and a pea protein.
 24. The process of claim 23,wherein the protein of interest comprises a heme protein and thecontinuous SMB extraction zone further comprises: a) the capture zonedisposed before the loading zone and after the equilibration zone andpassing at least a portion of the wash effluent stream to the top of thecapture zone and withdrawing a capture zone effluent from the bottom ofthe capture zone to minimize losses of the protein of interest in thewash zone and admixing the capture zone effluent with the first wastestream; b) a second regeneration zone disposed between the regenerationzone and the equilibration zone and counter currently or concurrentlypassing the second regeneration buffer stream comprising HCl to thesecond regeneration zone and withdrawing a second regeneration zoneeffluent stream and admixing the second regeneration effluent streamwith the second waste stream; and, c) a second equilibration zonedisposed between the second regeneration zone and the capture zone andpassing a second equilibration buffer stream comprising sodium orpotassium phosphate to the top of the second regeneration zone,withdrawing a second regeneration zone effluent and admixing the secondregeneration zone effluent with the third waste stream.